Highly sensitive platform to characterize extracellular vesicular biomarkers for cancer immunotherapy

Abstract

Methods and systems for characterizing extracellular vesicular biomarkers using a biochip with gold nanoparticles. The biochip includes a glass surface, a gold film layer on the glass surface, a plurality of gold nanoparticles coupled to the gold film layer, and a plurality of biotinylated antibodies coupled to the gold nanoparticles. In some implementations, the gold film layer of the biochip is coated with polyethylene glycol (PEG). The biotinylated antibodies are selected to capture specific types of extracellular vesicles. PD-L1/PD-1 proteins and RNAs in extracellular vesicles were characterized for cancer immunotherapy.

What is claimed is: 
     
         1 . A biochip for characterization of extracellular vesicular biomarkers for cancer immunotherapy, the biochip comprising:
 a glass surface;   a gold film layer on the glass surface;   a plurality of gold nanoparticles coupled to the gold film layer; and   a plurality of biotinylated antibodies coupled to the gold nanoparticles, wherein the biotinylated antibodies are selected to capture extracellular vesicles.   
     
     
         2 . The biochip of  claim 1 , wherein the gold film layer is coated with polyethylene glycol. 
     
     
         3 . The biochip of  claim 1 , wherein the gold film layer is coated with a linker solution including at least one selected from a group consisting of a 1-thiahexa(ethyleneoxide) lipidic anchor molecule WC14, a lateral spacer β-mercaptoethanol, and biotin-polyethylene glycol (PEG)-thiol (SH). 
     
     
         4 . The biochip of  claim 1 , wherein the gold film layer is coupled to the glass surface by a layer of (3-mercaptopropyl)trimethoxysilane (MPTMS). 
     
     
         5 . The biochip of  claim 1 , wherein the plurality of gold nanoparticles includes a plurality of streptavidin-conjugated gold nanoparticles. 
     
     
         6 . The biochip of  claim 1 , wherein each gold nanoparticle of the plurality of gold nanoparticles has a diameter of 30 nanometers. 
     
     
         7 . An assay platform system for identifying candidates for cancer immunotherapy, the system comprising:
 the biochip of  claim 1 ; and   a total internal reflection fluorescence (TIRF) microscope imaging system configured to capture image data of extracellular vesicular biomarker captured by the biochip.   
     
     
         8 . The system of  claim 7 , further comprising a controller configured to:
 detect and quantify the extracellular vesicular biomarker for a first patient captured by the biochip based at least in part on image data captured by the TIRF microscope imaging system;   compare a determined quantity of the extracellular vesicular biomarker to a threshold; and   identify the first patient as a candidate for cancer immunotherapy in response to determining that the determined quantity of the extracellular vesicular biomarker exceeds the threshold.   
     
     
         8 . A method of characterizing extracellular vesicular biomarkers for cancer immunotherapy, the method comprising:
 applying purified extracellular vesicles from a biofluid of a first patient to the biochip of  claim 1 ;   washing the biochip with a phosphate-buffered saline (PBS) solution;   applying a plurality of molecular beacons to the washed biochip;   rinsing the biochip with the PBS solution;   capturing image data of the biochip with a total internal reflection fluorescence (TIRF) microscope; and   detecting and quantifying occurrences of a first extracellular vesicular biomarker based on the captured image data.   
     
     
         9 . The method of  claim 8 , wherein applying the plurality of molecular beacons to the washed biochip includes applying a plurality of cationic lipoplex nanoparticles (CLN) containing PD-1/PD-L1 target-specific molecular beacons. 
     
     
         10 . The method of  claim 8 , further comprising:
 comparing a determined quantity of occurrences of the first extracellular vesicular biomarker to a threshold; and   determining whether the first patient is a candidate for cancer immunotherapy based at least in part on the comparison of the determined quantity of occurrences of the first extracellular vesicular biomarker and the threshold.   
     
     
         11 . A method for evaluating anti-PD-L1/PD-1 immunotherapy comprising coupling CD63/CD9 to a biochip of  claim 1  as the capture antibodies to capture EV-based PD-L1 and PD-1 mRNA and membrane proteins from a liquid biopsy sample. 
     
     
         12 . A method for evaluating an immunotherapy, the method comprising coupling at least one capture antibody to the biochip of  claim 1 , wherein the at least one capture antibody is selected to capture from a liquid biopsy sample at least one selected from a group consisting of a mRNA and a protein indicative of a response to the immunotherapy. 
     
     
         13 . A method for evaluating anti-PD-L1/PD-1 immunotherapy comprising using CD63/CD9 rich EV-based PD-L1 and PD-1 mRNA/membrane protein as a liquid biopsy biomarker for anti-PD-L1/PD-1 immunotherapy.